MTS assay.
The 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) cell cytotoxicity/proliferation assays were performed using the CellTiter 96 aqueous one-solution cell proliferation assay (Promega), according to manufacturer's instructions. For IL4 studies, splenocytes were cultured for 3 d in the presence or absence of 100 ng/ml IL4. Cells were seeded on day 3 in triplicate in a 96-well plate at 2.5 × 106 cells/ml at 100 μl per well. MTS reagent was added for 4 h and absorbance was read at 540 nm; values plotted were subtracted from blanks. For neutralization assays, splenocytes were seeded at 5 × 106 cells/ml at 100 μl per well on day of harvest and IL4, rat IgG1 anti-mouse IL4, or rat IgG1 isotype control (R&D Systems) were added at the concentrations indicated in the figures. Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding. For inhibitor studies, splenocytes were seeded at 1 × 107 cells/ml at 100 μl per well on day of harvest, and inhibitors were added at the concentrations indicated in the figures. The inhibitors BAY11–7085, rapamycin, triciribine, U0126, SB202190, and cucurbitacin I were purchased from EMD Biosciences. For non-malignant splenocyte cultures, B cell activation was induced with 10 μg/ml of goat F(ab') anti-mouse IgM (Jackson ImmunoResearch). Cultures were pulsed for 4 h with MTS reagent 1 d post-seeding.